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Best proof-reading polymerase?


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12 replies to this topic

#1 Trof

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Posted 06 November 2012 - 03:17 AM

Since we already agreed elsewhere that best Taq polymerase is QIAGENs HotStarTaq (or actually their reaction buffer is) I'm now looking for comparably good easy-to-use proofreeding polymerase, that I would use on special occasions.

I heard repeatedly good things about Phusion from NEB. Since I want a master mix and hot-start version (preferably) I was looking at Phusion Hot Start Flex 2X Master Mix.

If you have other suggestions for good proof-read polymerase, please share them. Thanks.

I would just like to point out that master mix option and hot start version is definitely prefered (if not for anything for the fact we don't have separate PCR components anymore).

Our country has a serious deficiency in lighthouses. I assume the main reason is that we have no sea.

I never trust anything that can't be doubted.

'Normal' is a dryer setting. - Elizabeth Moon


#2 leelee

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Posted 06 November 2012 - 03:46 AM

I Posted Image Phusion

(I know you said other suggestions, but figured another vote for Phusion can't be a bad thing Posted Image )

#3 hobglobin

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Posted 06 November 2012 - 09:29 AM

is there really a best one (as with Taq polymerase)? They work at all or even best for a specific PCR or not (after trial and error), and for an unproblematic PCR the cheapest is the best Posted Image
One must presume that long and short arguments contribute to the same end. - Epicurus
...except casandra's that belong to the funniest, most interesting and imaginative (or over-imaginative?) ones, I suppose.

#4 bob1

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Posted 06 November 2012 - 11:37 AM

I've had good times with KOD polymerase and Pwo. KOD especially has a very high processivity, which makes for fast PCRs.

#5 Trof

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Posted 06 November 2012 - 11:40 AM

hobglobin@
Well the best one is such, that has best sucess rate on all posible templates (but of course if I have a GC rich one or some other speciality I need to consider this) so I don't need to lose time with trying which one will work this time.
HSTaq is definitelly not cheapest, but it never failed me and I never lost much time in optimising (mostly I didn't lose any time at all, since it just worked the first time). I'm willing to pay for this, because more and more time becomes the most valuable thing ;)

Our country has a serious deficiency in lighthouses. I assume the main reason is that we have no sea.

I never trust anything that can't be doubted.

'Normal' is a dryer setting. - Elizabeth Moon


#6 Mad researcher

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Posted 06 November 2012 - 01:34 PM

Try Sigma Aldrich Jump start Taq DNA
Cheers,

Mad Researcher

#7 Trof

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Posted 06 November 2012 - 02:06 PM

Try Sigma Aldrich Jump start Taq DNA

I'm affraid that from the fact that it's a Taq, it's not proof-reading.

Our country has a serious deficiency in lighthouses. I assume the main reason is that we have no sea.

I never trust anything that can't be doubted.

'Normal' is a dryer setting. - Elizabeth Moon


#8 ascacioc

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Posted 06 November 2012 - 03:57 PM

I agree with leelee and another Phusion vote gets here; BTW, for the people who want their own Phusion: clone Pfu behind a single strand binding domain from a thermophile and then you get Phusion in your lab and it expresses like hell with a His tag in the classical way. From 1L, in one His column purification step you get enough for your entire institute forever and ever.

Andreea

#9 Trof

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Posted 07 November 2012 - 04:07 AM

Did I tell you before kind of I love your polymerase hacking? ;)

Our country has a serious deficiency in lighthouses. I assume the main reason is that we have no sea.

I never trust anything that can't be doubted.

'Normal' is a dryer setting. - Elizabeth Moon


#10 hobglobin

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Posted 07 November 2012 - 09:30 AM

hobglobin@
Well the best one is such, that has best sucess rate on all posible templates (but of course if I have a GC rich one or some other speciality I need to consider this) so I don't need to lose time with trying which one will work this time.
HSTaq is definitelly not cheapest, but it never failed me and I never lost much time in optimising (mostly I didn't lose any time at all, since it just worked the first time). I'm willing to pay for this, because more and more time becomes the most valuable thing Posted Image

I used this Taq also a lot but with my last samples from a new insect species (amplifying microsatellite loci) it failed and now the cheap Bioline taq gives good and consistent results...
So as I'm working on several different species and have not just human samples and a known sequence, I've to try out and some polymerases work, some not. So for me it's often trial and error and there is no best.
Anyway we also should try out to make our own Phusion...Posted Image

Edited by hobglobin, 07 November 2012 - 09:32 AM.

One must presume that long and short arguments contribute to the same end. - Epicurus
...except casandra's that belong to the funniest, most interesting and imaginative (or over-imaginative?) ones, I suppose.

#11 Trof

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Posted 07 November 2012 - 10:00 AM

I used this Taq also a lot but with my last samples from a new insect species (amplifying microsatellite loci) it failed and now the cheap Bioline taq gives good and consistent results...
So as I'm working on several different species and have not just human samples and a known sequence, I've to try out and some polymerases work, some not. So for me it's often trial and error and there is no best.
Anyway we also should try out to make our own Phusion...Posted Image

For sure they optimized their mix for certain application and human/mammalian is the most common. So I wouldn't be surprised that special application have special requirements. Also many people who work with enviromental samples use rather a inhibitor resistant polymerase or something.
And as for Phusion, I like the idea but again, if I can afford it, I'm pretty much willing to pay a company for their good polymerase they spend years creating.

Our country has a serious deficiency in lighthouses. I assume the main reason is that we have no sea.

I never trust anything that can't be doubted.

'Normal' is a dryer setting. - Elizabeth Moon


#12 phage434

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Posted 11 November 2012 - 07:49 AM

Phusion works very well, but I've found that the new NEB Q5 polymerase also is very good. I'm trying to see which I like better, but Q5 is currently leading.

#13 badguy

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Posted 17 December 2012 - 09:28 PM

I read from Biotechniques, it says that Phusion from Finnzymes has the best proof reading activity.




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